Immune modulation by mesenchymal stem cells

Abstract Mesenchymal stem cells (MSCs) can be derived from various adult tissues with multipotent and self‐renewal abilities. The characteristics of presenting no major ethical concerns, having low immunogenicity and possessing immune modulation functions make MSCs promising candidates for stem cell therapies. MSCs could promote inflammation when the immune system is underactivated and restrain inflammation when the immune system is overactivated to avoid self‐overattack. These cells express many immune suppressors to switch them from a pro‐inflammatory phenotype to an anti‐inflammatory phenotype, resulting in immune effector cell suppression and immune suppressor cell activation. We would discuss the mechanisms governing the immune modulation function of these cells in this review, especially the immune‐suppressive effects of MSCs.


| INTRODUC TI ON
Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells, are spindle-shaped cells with multipotent (chondrocyte, osteoblast and adipocyte) and self-renewal abilities. 1,2 These cells are derived from various adult tissues, 3,4 attach to tissue culture dishes and express certain cell surface markers (positive for CD73, CD90 and CD105; negative for CD45, CD34, CD14 or CD11b, CD79alpha or CD19, and HLA-DR). 2 MSCs can be safely harvested with no major ethical concerns and have low immunogenicity. 3 Therefore, MSCs have been proposed as effective and safe cell sources for stem cell therapy.
Although MSCs have differentiation abilities, the main mechanism of their therapeutic effects in pre-clinical and clinical studies is believed to be paracrine effects. These paracrine effects include promoting angiogenesis, preventing apoptosis, suppressing inflammation and modulating extracellular matrix dynamics. One of the ways that these cells improve the tissue microenvironments is by modulating immune system components, such as macrophages and neutrophils. After the tissues or cells are injured, the MSCs activate or suppress the immune system to control the whole-tissue regeneration process. [3][4][5][6] Mesenchymal stem cells have been successfully applied in treating various diseases such as diabetes, 7 cardiovascular diseases, 8 graft-versus-host diseases 9 and autoimmune diseases. 10 Although many questions remain unanswered the immune modulation effects of MSCs make them promising candidates for cell therapy-based tissue repair and disease treatment, especially for immune system abnormalities, such as cancer and autoimmune diseases. Thus, we will discuss the mechanisms of immune modulation by MSCs. Given the important roles of MSCs in immune suppression to help cancer to escape immune surveillance and their potential roles in immune tolerance re-establishment, we mainly focus on the immune-suppressive function of MSCs in the current review.

| IMMUNE MODUL ATI ON BY MSC S
Mesenchymal stem cells could promote inflammation when the immune system is underactivated and restrain inflammation while the immune system is overactivated to avoid self-overattack. This activity is also known as the function of "sensor and switcher of the immune system" (Figure 1). 11 The MSCs could sense different danger signals through TLRs (Toll-like receptors). [12][13][14][15][16] MSCs express TLR2, TLR3, TLR4, TLR7 and TLR9. The expression levels of these TLRs vary significantly based on their tissue origin. 17 TLRs recognize molecules from injured cells or pathogens acting as the first line of the immune defence system. TLR activation can further stimulate immune cells and MSCs. 17 Activated MSCs respond to TLR ligands and release anti-inflammatory factors. Thus, TLRs play an important role in sensing and switching immune responses by MSCs. 17 The allogeneic MSCs would be eliminated by NK cells slowly. However, once the MSCs are activated via TLR3 ligand, they could escape from this clearance process by NK cells. 18 The type of TLR (TLR3 or TLR4) activation could also induce a pro-inflammatory or anti-inflammatory phenotype of MSCs. [12][13][14] For example, TLR3 activation induces an anti-inflammatory phenotype of MSCs (also known as the MSC2 phenotype), while TLR4 activation induces a pro-inflammatory phenotype (also known as the MSC1 phenotype). 3,14 Furthermore, the MSC microenvironment could switch the MSCs between pro-inflammatory and anti-inflammatory phenotypes.
MSCs have pro-inflammatory functions in the early stage of inflammation through recruiting neutrophils. 19 Pro-inflammatory MSCs activate T cells by secreting MIP-1 (macrophage inflammatory protein-1), CCL5 (C-C motif ligand 5), CXCL9 (C-X-C motif ligand 9) and CXCL10 (C-X-C motif ligand 10) and recruiting more lymphocytes. 3 At this stage, there are only low levels of inflammation signals, such as TNF-α and IFN-λ. MSCs derived from bone marrow and umbilical cord promote immune response when they are treated with low levels of IFN-γ and TNF-α, which could not produce sufficient iNOS or IDO to suppress the lymphocytes. 20 However, when these two cytokines reach a high level, they stimulate MSCs to secrete iNOS (mice) or IDO (human), resulting in T-cell proliferation inhibition and Treg induction. Therefore, the iNOS or IDO level has been proposed as the switcher between the pro-and anti-inflammatory effects of MSCs. 3 TNF-α and IFN-λ are often used for MSC activation. 21  It has been demonstrated that MSCs also represent one type of cell to prevent overstimulation of the immune system. The immune-suppressive activities of MSCs are primarily stimulated by pro-inflammatory factors, such as IFN-γ (interferon gamma), TNF-α (tumour necrosis factor alpha) and IL-1β (interleukin-1 beta). [4][5][6] Among these factors, IFN-γ is even more crucial for the immune-

| IDO
IDO has two isoforms, IDO1 and IDO2. These isoforms catalyse tryptophan, an important essential amino acid, into different metabolites, resulting in tryptophan depletion. 29 Because tryptophan is essential for T-cell proliferation, 30 tryptophan depletion switches the metabolic pathway from glycolysis to oxidative phosphorylation, resulting in T-cell arrest. 31 Tryptophan reduction also induces the accumulation of uncharged tryptophan tRNA in immune cells, which could activate stress-response kinase GCN2 (general control nonderepressible 2) and eIF2 (eukaryotic translation initiation factor 2)-mediated pathways, leading to protein synthesis reduction, cell proliferation inhibition and Fas-mediated lymphocyte apoptosis. 32 GCN2 pathway activation also promotes Treg differentiation while suppressing Th17 conversion through downregulating IL-6. 33 Tryptophan deprivation could induce Treg generation through producing tolerogenic DCs, with downregulation of co-stimulatory molecules and upregulation of the inhibitory receptors ILT3 (immunoglobulin-like transcript 3) and ILT4 (immunoglobulin-like transcript 4) on DCs. 34 The tryptophan metabolites (kynurenine, quinolinic acid and picolinic acid) are more toxic to CD4 + Th1 and CD8 + T cells and less toxic to Th2 cells, thereby switching T helper cells from Th1 to Th2. 35 Furthermore, the tryptophan metabolite kynurenine could directly bind to AhR (aryl hydrocarbon receptor) and promote CD4 + Foxp3 + Treg differentiation while suppressing Th17 generation and decreasing DC immunogenicity. 36 IDO is primarily expressed by antigen-presenting cells. 32 MSCs also express and utilize IDO to mediate immune suppression. 37

| PGE2
PGE2 is produced by COX-1 (cyclooxygenase-1, the constitutive isoform) or COX-2 (cyclooxygenase-2, the inducible isoform) from the arachidonic acid released from the membrane phospholipids. PGE2 interacts with EP2 and EP4 receptors expressed on the surface of immune cells and exerts its anti-inflammatory effects. The interaction between PGE2 and EP2 or EP4 receptors induces cyclic AMP (cAMP) upregulation, which then activates the PKA (protein kinase A) and PI3K (phosphatidylinositol-3 kinase) pathways. cAMP induces the expression of anti-inflammatory factors (IL-4, IL-5 and IL-10) and inhibits the expression of pro-inflammatory factors (IL-12p70, TNF-α, CCL3 and CCL4) through IL-2 pathway suppression. In addition, cAMP promoted M2 macrophage and Th2 cell differentiation and inhibited Th1 production. [40][41][42] However, some studies have shown that PGE2 has pro-inflammatory effects with enhancing DC maturation and T-cell proliferation. 43 Later studies have demonstrated that a low concentration of PGE2 promotes an inflammatory response, while a high concentration inhibits. 43 PGE2 promotes Foxp3 + Treg cell production. 44 PGE2 also promotes TGF-β secretion from monocytes and induces MDSC (myeloid-derived suppressor cells) generation, which could suppress NK cell and CD8 + T-cell activities. 45,46 PGE2 suppresses IL-12 and promotes IL-23 expression. IL-12 (IL-12p70) is composed of IL-12p35 and IL-12p40. The suppression of IL-12 by PGE2 is mediated through inhibiting IL-12p35 but not IL-12p40. PGE2 could increase IL-23p19 expression, which could form IL-23 with IL-12p40. Thus, PGE2 induces IL-23 expression, which is important for Th17 production. 47,48 MSCs express COX-2 and produce PGE2, 11,49 which could be further enhanced by inflammatory stimuli or the combination of IFN-γ and TNF-α treatment. 50 Therefore, these cells produce high amounts of PGE2 to suppress the immune response. 51

| iNOS
Mesenchymal stem cells express iNOS, which metabolizes L-arginine to generate NO (nitric oxide). 37,52 NO suppresses the IL-2 pathways (Janus kinase 3, signal transducer and activator of transcription 5, extracellular signal-regulated kinases and protein kinase B), resulting in T-cell proliferation and function inhibition. [52][53][54][55] NO also induces T-cell apoptosis and inhibits the expression of MHC-II. 56 NO suppresses the secretion of Th1 and Th2 cytokines. 57,58 When MSCs are stimulated with inflammatory factors, the iNOS gene is upregulated. These cells produce high amounts of NO to suppress the immune response. 21,51 Interestingly, the pro-inflammatory cytokine IL-17 could stabilize the iNOS protein in MSCs derived from bone marrow, resulting in immune suppression. 59 MSCs from mice, rabbits, rats and hamsters mainly exert suppressive functions through iNOS, while MSCs derived from humans, pigs and monkeys primarily exert suppressive functions through IDO. 60 Thus, the mechanism of immune-suppressive functions of MSCs from different species might differ in the detailed pathways.

| TGF-β
TGF-β and IL-10 are the main immune-regulatory cytokines generated by quiescent MSCs. 61,62 TGF-β is constitutively secreted by MSCs 63 and further upregulated by inflammatory factors, such as IFN-γ and TNF-α. 50,64,65 TGF-β inhibits IL-2, MHC-II (major histocompatibility complex II) and co-stimulatory factor expression in DCs and T cells. 61,62 Both Th1 differentiation and Th2 differentiation could be inhibited by TGF-β. 66,67 TGF-β promotes Treg and Breg production. 61 TGF-β is one of the key regulators of Foxp3 expression. 61,62 However, it has also been shown that the immune suppression effects of bone marrow-derived MSCs stimulated with IFN-γ and TNF-α are abolished by adding TGF-β through inhibiting iNOS and IDO expression. 68

| IL-10
In addition to TGF-β, IL-10 is another main immune-suppressive cytokine generated by quiescent MSCs. IL-10 expression could be further enhanced by TLR ligands and PEG2. 69

| CD39 and CD73
MSCs express CD39 and CD73. CD39 catabolizes ATP to AMP, and CD73 catabolizes AMP to adenosine. Extracellular ATP has pro-inflammatory effects, while adenosine has anti-inflammatory effects through the cAMP and PKA pathways. Thus, CD39 and CD73 could cleave extracellular ATP to adenosine and switch pro-inflammation to anti-inflammation. 83,84

| CCL2
Mesenchymal stem cells express CCL2 and the related metalloproteinases that are responsible for CCL2 cleavage. The truncated CCL2 functions as a CCR2 antagonist and inhibits immune cell migration.
While the full-length CCL2 binds to its receptor CCR2, which is expressed by activated Th1, Th17 and NK cells, and recruits them into the inflammation sites, the truncated CCL2 plays a critical role in the autoimmunity suppression by MSCs.

| HO-1
Both human and rat MSCs express a high level of HO-1 in the quiescent state. 96 Blocking HO-1 reduced the immune-suppressive effects of MSCs. 96 HO-1 could promote IL10 + Tr1 and TGFβ + Tr3 generation, two types of Treg. 97 However, once MSCs are activated by pro-inflammatory factors, HO-1 expression decreases rapidly, and the immune-suppressive function of MSCs is taken over by other suppressive factors, such as iNOS. 97

| TSG6
The aggregated MSCs and MSC spheres express TSG6, an important immune-suppressive factor. 98,99 TSG6 could reduce lymphocyte and neutrophil proliferation and decrease metalloproteinase activity and the expression of IL-6 and IFN-γ. On the other hand, TSG6 could promote Foxp3 + Treg and IL10 + iNOS + regulatory macrophage expansion. 98

| IL1RA
IL1RA expressed by MSCs could promote M2 macrophage polarization and Treg generation with elevated IL-10 expression and suppress CD4 + T-cell activities. Furthermore, IL1RA could suppress B-cell differentiation and antibody production. 100,101

| Complement system-related proteins
MSCs express C3aR (C3a receptor) and C5aR (C5a receptor), which could be activated by C3a and C5a produced in the inflammation sites. The activated C3aR/C5aR could enhance the resistance to oxidative stress and apoptosis of MSCs. 102 On the other hand, CD46, CD55 and CD59 expressed on the surface of MSCs could inhibit complement system activation and prevent MSCs from cell lysis. 102,103 However, once cell lysis is activated by the complementary system, this protection is not sufficient to stop the cell death process. 104 Combining IFN-γ treatment with TNF-α significantly increases the ability of MSCs to secrete factor H, which is a key molecule involved in the inhibition of complement activation. 103

| DC
Mesenchymal stem cells suppress DC differentiation, maturation and activation and compromise their antigen presentation abilities. [105][106][107][108][109] MSCs inhibit DC differentiation from monocytes or CD34 + HSCs (hematopoietic stem cells), resulting in immature DC production and immune suppression. 105,110 MSCs could downregulate the expression of HLA II, CD80, CD86 and IL-12 in DCs, resulting in the inhibition of DC maturation and activation. [110][111][112] For mature DCs, MSCs could inhibit DC migration by downregulating CCR7 and CD49dβ1 and decreasing inflammatory factor expression and antigen presentation abilities. 109,[113][114][115] MSCs could switch the mature DCs into a suppressive immature phenotype through the Jagged1 or IL-10-SOCS3 pathway. [116][117][118] MSCs promote IL-10-positive pDCs (plasmacytoid DCs) differentiation, 11 which would promote Treg development. 119 Mesenchymal stem cells could also induce DCs into an anti-inflammatory phenotype through downregulating the pro-inflammatory factors (TNF-α and IL-12) and upregulating the anti-inflammatory factors, such as IL-10, 11,120 PGE2 121,122 and M-CSF (macrophage colony-stimulating factor). 123,124 MSCs induce DCs to secrete IL-10 and then inhibit T-cell activation. 11,120 MSCs also express high levels of PGE2, which binds to its receptor EP4 on DCs and exerts inhibitory effects. 121,122 The direct contact between MSCs and DCs activates the Notch pathway and suppresses DC generation 106 and proliferation. 125 Furthermore, MSCs block the cell interaction between DCs and lymphocytes. 126 On the other hand, DCs support MSC survival through lymphotoxin-β expression. 127

| Monocytes/Macrophages
Monocyte modulation is the critical step in the immune modulation process, as depleting monocytes would abolish the immune-suppressive effects of MSCs. 128,129 These results support the hypothesis that the immune-suppressive effects of MSCs are mainly induced through monocyte/macrophage modulation by MSCs.

| Regulatory B cells
Mesenchymal stem cells could promote Breg production (CD19 + CD24 high CD38 high in humans and CD19 + CD1d high CD5 + in mice) with IL-10 expression. 9,101,148,154,159,160 The expansion of Breg cells promoted by MSCs might account for the total B-cell population expansion in some studies. 152,161

| Th1
Mesenchymal stem cells exert immune-suppressive effects through inhibiting Th1 type pro-inflammatory factor expression (such as IFN-γ, TNF-α and IL-1β) and enhancing Th2 type factor expression. 11 MSCs promote Th1 cells to secrete the immune suppressor IL-10 and thus repress the immune responses. 193 MSCs also inhibit Th1 cell activation indirectly through suppressing DC and NK cells. 113

| Th2
Mesenchymal stem cells induce the differentiation and maturation of Th2 cells through IDO expression, which causes tryptophan depletion and tryptophan metabolite production. 194 The tryptophan metabolites also induced Th1 cell apoptosis. 195

| ILC
Mesenchymal stem cells support the differentiation of ILC2 (group 2 innate lymphoid cells), 224 and the expansion and activity of ILC3 (group 3 innate lymphoid cells). 225 Furthermore, ILC3 also promotes the activity of MSCs. 225 Both ILC2 and ILC3 have tissue-protective functions, such as anti-inflammation and promoting tissue regeneration. 226,227 Recently, it has been demonstrated that ILC3 could support and generate Treg cells by secreting IL-2. 228

| Neutrophils
MSCs suppress neutrophil recruitment, activation, extracellular trap formation and protease secretion by secreting superoxide dismutase-3. 229-231 However, some reports have shown that MSCs protect neutrophils from apoptosis, promote their function through the IL-6 and STAT3 pathways, [232][233][234] and promote neutrophil recruitment through IL-8 and MIF (macrophage migration inhibitory factor) secreted by MSCs. 235 This feature is correlated with the pro-inflammation phenotype of MSCs. 12,107,236,237 Thus, neutrophil modulation by MSCs might also depend on the pro-inflammatory or anti-inflammatory phenotype of MSCs.

| CON CLUS ION
Although further efforts should be made to understand the biological

CO N FLI C T O F I NTE R E S T
The authors declare no commercial or financial conflict of interest.

AUTH O R CO NTR I B UTI O N
WJ collected the updated references; JX wrote the manuscript and draw the figures.

FUTU R E PE R S PEC TI V E S
Since the first demonstration of MSCs, many achievements have been made to understand their localization, function and underlying mechanisms. However, many unresolved issues still need to be addressed. In this section, we would like to raise three main questions that should be answered in the coming future.
First, what is the specific cell marker for MSCs? Several MSC markers have been demonstrated, but none of them are specific. 243